The goal of the proposed study is to examine the role of endothelin-A (ETA) receptor signaling during cephalic neural crest cell development. Our preliminary results indicate that ETA receptor signaling is likely required for multiple aspects of neural crest development. We hypothesize that ETA receptor signaling is required by a subset of cephalic neural crest cells for both their migration into the arches and subsequent patterning of specific arch subregions during craniofacial development. Mice homozygous for a targeted mutation in the ETA gene are born with numerous craniofacial birth defects resulting from aberrant neural crest development. Our preliminary data show that loss of ETA signaling 1) results in absence of ETA-/- cells in the distal pharyngeal arches of ETA-/- +1+ chimeras, 2) prevents normal post-migratory neural crest cell proliferation and 3) disrupts development of neural crest-derived mesenchymal cells within the pharyngeal arches, but only in a region-specific manner. We have three specific hypotheses: 1) ETA receptor signaling is required for both cephalic crest cell migration into the pharyngeal arches and post-migration proliferation, 2) This dual action represents two distinct requirements for ETA signaling and 3) ETA expression in different arch subregions initiates or maintains distinct signaling cascades involved in the development of specific craniofacial structures. Our planned experiments include elucidating the fate of ETA-/- neural crest cells by following their migration and proliferation using a genetic marking system. We will investigate the timing of ETA action during neural crest development by transient blockade of ETA signaling in pregnant mice, using a potent and specific ETA receptor antagonist. Finally, we will investigate the regional significance of ETA receptor signaling within the first mandibular arch by inactivating the ETA gene in arch subregions using Cre/loxP technology. Understanding the mechanisms by which ETA receptor signaling contributes to cephalic neural crest cell development will be an important advance in understanding the genetic and cellular control of craniofacial development and the etiology leading to specific human craniofacial birth defect syndromes.